Heparin, a substance discovered by McLean at the beginning of the XX century, is applied in clinics since 1937 and is the first polysaccharide-based drug included in the WHO list of essential medicines. Heparin is a glycosaminoglycan (GAG) with a large molecular weight dispersity and high degree of substitution with sulfate groups (it has the largest density of a negative charge among biomolecules reaching 2.7 charges per a repeat unit consisting of a glucosamine group and an L-iduronic acid group). Heparin is produced and stored in mast cells, macrophages and in the vascular endothelium. It is obtained from the animal tissues, particularly from porcine intestines and bovine lungs. It shows very strong inhibition of blood coagulation, although only one third of heparin molecules show anticoagulative properties. Its action is based on enhancing the ability of antithrombin (AT) to deactivate thrombin and Xa factor, the enzymes responsible for the production of fibrin during clot formation. Heparin is a drug of choice in the situations when fast inhibition of coagulation is necessary, e.g. during surgical operations, in particular to prevent formation of clots in the apparatus used in extracorporeal circulation, such as dialyzers or oxygenators. It also has many other therapeutic applications, e.g., in the treatment of unstable angina pectoris or acute heart infarction. It may also lower cholesterol and lipids. Two laboratory tests are used to assess the activity of heparin, i.e., activated partial thromboplastin time (aPTT) and activated clotting time (ACT). The former is used for more precise monitoring of lower doses and is applied rather in prophylaxis while the latter is used to monitor general heparin activity in a wide range of doses and is used during surgical operations.
However, the application of heparin is connected with many adverse effects, among which the most frequent ones are hemorrhages, heparin induced thrombocytopenia (HIT), hypercalcemia which results in osteoporosis on prolonged administration of heparin and increased aminotransferase level in blood. In order to prevent the adverse effects of lowered coagulation it is often necessary to neutralize or remove heparin from blood after an expected anticoagulation effect is reached. Also, because of the adverse effects of heparin, low molecular weight heparins (LMWH) are more and more frequently used, which due to shorter chains show mainly anti-Xa activity. Due to the differences in the mechanism of action these drugs are safer and show prolonged action therefore they may be chronically applied. Their drawback is that they have no effective antidote and when overdosed it is more difficult to return normal blood coagulation in a patient. When it is necessary to assess their anticoagulative action, the activity against the active Xa factor (anti-Xa activity) is evaluated.
In the current state of art the only clinically used drug neutralizing heparin action is protamine, a protein which was introduced to clinical application almost simultaneously with heparin (Fischer, A Biochem Zeit. 278, 133, 1935.). It possesses exceptionally high content of basic amino acids such as arginine, lysine, and histidine, which may reach 80%. Another polymer which was studied for heparin removal is poly-L-lysine (Ma, X., Mohammad, S. F., Kim, S. W., Biotechnology and Bioengineering, 1992, 40(4), 530-536).
Yet another approach to heparin neutralization problem is its enzymatic degradation using immobilized heparinase (Kolde, H.-J., Pelzer, H., Borzhenskaya, L., Russo, A., Rose, M., Tejidor, L. Hamostaseologie 1994, 14(1), 37-43).
Unfortunately, the mentioned methods of heparin neutralization may have adverse effects. Protamine, if not neutralized or removed from blood, may result in adverse reactions in about 10% of patients. They may be very serious and even lethal including pulmonary hypertension, arterial hypotension, anaphylactic shock, thrombocytopenia and granulocytopenia. Heparin neutralization with protamine is incomplete and is accompanied with allergic reactions.
Anticoagulative activity of low-molecular-weight heparins may be only partially neutralized (up to 60% maximum) by intravenous injection of protamine sulfate (http://products.sanofi.us/lovenox/lovenox.html). However, except for protamine there are no other compounds neutralizing these anticoagulants on the market. The introduction of a safe and efficient antidote for low-molecular-weight heparins could extend their applications with those currently typical of unfractionated heparin.
Except for chemical methods of heparin neutralization, there were also studies on the methods of its physical removal from blood.
The devices for physical heparin removal from blood were mostly based on the application of immobilized poly-L-lysine (Joseph B. Zwischenberger, MD, Roger A. Vertrees, BA, CCP, Robert L. Brunston, Jr., MD, Weike Tao, MD, Scott K. Alpard, MD, and Paul S. Brown, Jr., MD, The Journal of Thoracic and Cardiovascular Surgery 1998 Volume 115, Number 3; Zwischenberger, J. B., Tao, W., Deyo, D. J., Vertrees, R. A., Alpard, S. K., Shulman, G. Annals of Thoracic Burgery Tom 71, Issue 1, 2001, Pages 270-277). The heparin removal device (HRD) described in the above papers, was included into the blood circulation system of a patient by extracorporeal venous-venous shunt. It allowed separation of serum, which upon heparin removal through the contact with poly-L-lysine, was returned into the patient's blood. In spite of promising results the experiments with the application of the devices of this type are limited and so far none of them has been introduced into clinical practice.
The method frequently used in order to avoid complications resulting from unbound heparin antagonists is their immobilization on polymeric supports in the heparin removal device. For example, protamine was supported on a matrix obtained by grafting an acrylic polymer onto cellulose (Hou, K. C., Roy, S., Zaniewski, R., Shumway, E. Artificial Organs 1990, 14((6), 436-442) or inside cellulose fibres (Wang, T., Byun, Y., Kim, J.-S., Liang, J., Yang, V. C. International Journal of Bio-Chromatography 2001, 6(2), 133-149). It was shown that the bioreactor removed more than 50% of administered heparin during 10 at the blood flow of 100 mL/min. The application of a bioreactor containing immobilized protamine did not result in any statistically significant changes in the monitored hemodynamic parameters.
Another paper reports efficient heparin removal using spheres obtained from alginate and poly-L-lysine (M. Sunil Varghese, D. Hildebrandt, and D. Glasser, N. J. Crowther, D. M. Rubin, Artificial Cells, Blood Substitutes, and Biotechnology, 2006, 34, 419-432). However, the polymeric spheres could not be applied in vivo.
Inventors of the present invention have previously developed heparin neutralization methods based on the application of polysaccharides in the soluble form to neutralize heparin without its physical removal from blood (patent application P 387249) or based on crosslinked polysaccharides in the form of microspheres for the neutralization of heparin in blood by its physical removal. The studies on antiheparin activity of polymers described in the present application are a continuation of the studies described in the previous applications.
The methods described above do not allow effective heparin neutralization. Protamine used commonly in clinical practice may cause serious adverse effects, while the attempts to remove heparin from blood with the methods of its physical removal were found to be uncomfortable and impractical due to the limitations in their application, including the necessity of hospitalization of the patients.